Pharmaceutical compositions of 5-alkynyl-dideoxyribouracil

ABSTRACT

This application relates to pharmaceutical compositions of 5-alkynyl-dideoxyridbouracils which generates 5-alkynyluracils in vivo, which are inactivators of uracil reductase.

This application is a continuation of Ser. No. 08/336,717, filed Nov. 9,1994, now U.S. Pat. No. 5,643,913 which is a continuation of Ser. No.07/965,261, filed Jan. 19, 1993, now abandoned, which is a 371 ofPCT/GB91/01197 filed Jul. 18, 1991.

The present invention relates to certain enzyme inactivators which areespecially useful for co-administration with other therapeutic compoundssuch as antiviral compounds in order to provide an improved therapeuticindex by reducing the toxic side-effects.

A therapeutic nucleoside analogue that has been found to have aparticularly beneficial clinical effect against a spectrum of conditionsassociated with Human Immunodeficiency Virus (HIV) infections such asAcquired Immune Deficiency Syndrome (AIDS), AIDS-related complex (ARC)and asymptotomatic infections, is the compound3′-azido-3′-deoxythymidine having the approved name zidovudine. Thiscompound at low doses is generally very well tolerated by patients andis now widely used in the treatment of HIV infections. However, incertain patients treated with zidovudine, some haematogical suppressionincluding anaemia and neutropenia may be observed, presumably arisingfrom a certain limited level of toxicity of zidovudine observed towardsstem cells. Other less commonly observed side-effects have beendescribed such as myopathy which may be related to intracellularactivity of zidovudine.

It has now been found that the stem cell and haematological toxicity ofzidovudine can be reduced by co-administration of an inactivator of theenzyme uracil reductase (dihydropyrimidine dehydrogenase, EC 1.3.1.2)which reduces the degradation of uracil.

The present invention is thus based on the discovery that the use of aninactivator of uracil reductase in combination with zidovudine reducesthe cellular toxicity of zidovudine.

According to the present invention therefore we provide a uracilreductase inactivator for use in medical therapy, especially incombination with zidovudine or a pharmaceutically acceptable salt orester thereof, for example in the treatment or prophylaxis of HIVinfections such as AIDS, ARC and asymptomatic infections.

The present invention further provides:

a) a combination of a uracil reductase inactivator and zidovudine or apharmaceutically acceptable salt or ester thereof;

b) a method for the treatment or prophylaxis of an HIV infection in ahuman which comprises administering to the said human an effectiveanti-HIV amount of zidovudine or a pharmaceutically acceptable salt orester thereof in combination with a uracil reductase inactivator.

It should be noted that the references herein to uracil reductaseinactivators refer to compounds that inactivate the uracil reductaseenzyme, effectively acting as suicide substrates, in contrast tocompounds that merely have an inhibiting effect on the enzyme.

It has been found that particularly beneficial effects in reducing thetoxicity of zidovudine have been achieved using as a uracil reductaseinactivator a 5-substituted uracil compound, particularly a uracilcompound substituted in the 5-position by a halogen atom e.g. iodine orbromine; a C₂₋₄ alkenyl group (e.g. vinyl) optionally substituted byhalogen e.g. 2-bromovinyl, 1-chlorovinyl or 2-bromo-1-chlorovinyl; aC₂₋₆ alkynyl group optionally substituted by a halogen (e.g. bromine)atom; a cyano group; or a C₁₋₄ alkyl group substituted by halogen e.g.trifluoromethyl. Particularly preferred inactivators of uracil reductasefor use in accordance with the invention are 5-ethynyluracil and5-propynyluracil. Other inactivators for such use include:

5-ethynyluracil

5-cyanouracil

5-propynyluracil

5-bromoethynyluracil

5-(1-chlorovinyl)uracil

5-iodouracil

5-bromovinyluracil

5-hex-1-ynyluracil

5-vinyluracil

5-trifluorouracil

5-bromouracil

5-(2-bromo-1-chlorovinyl)uracil

In experiments in mice, it has been found that red-blood cell anaemiainduced by treatment with zidovudine could be at least partiallyprevented by treatment with 5-ethynyluracil.

The above 5-propynyluracil is a novel compound and represents a furtherfeature of the present invention.

Other uracil reductase inactivators which may be employed in accordancewith the present invention include compounds which generate the aboveuracil compounds in vivo. Such compounds include nucleoside derivativeswhich contain a nucleobase corresponding to the above 5-substituteduracil compounds, for example nucleoside derivatives containing aribose, 2′-deoxyribose, 2′,3′-dideoxyribose, arabinose or othercleavable sugar portion, which may additionally contain a 2′- or3′-substituent such as halo, for example fluoro. Specific examples ofsuch nucleoside derivatives are1-(β-D-arabinofuranosyl)-5-prop-1-ynyluracil and2′,3′-dideoxy-5-ethynyl-3′-fluorouridine.

Zidovudine or a pharmaceutically acceptable salt or ester thereof andthe said uracil reductase inactivator may be employed in combination inaccordance with the invention by administration of the components of thecombination to an appropriate subject either concomitantly, for examplein a unitary pharmaceutical formulation, or, more preferably,separately, or sequentially within a sufficient time period whereby thedesired therapeutic effect of the combination is achieved.

Zidovudine or a pharmaceutically acceptable salt or ester thereof andthe uracil reductase inactivator may be administered respectively fortherapy by any suitable route including oral, rectal, nasal, topical(including buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal); the oralroute is especially preferred. It will be appreciated that the preferredroute will vary with the condition and age of the recipient, the natureof the infection and other clinical factors.

In general a suitable dose of zidovudine or a pharmaceuticallyacceptable salt or ester thereof will be in the range of 1.0 to 120 mgper kilogram body weight of the recipient per day, preferably in therange of 2 to 30 mg per kilogram body weight per day and most preferablyin the range of 5 to 20 mg per kilogram body weight per day. The desireddose is preferably presented as two, three, four, five, six or moresub-doses administered at appropriate intervals throught the day. Thesesub-doses may be administered in unit dosage forms, for example,containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably50 to 700 mg of active ingredient per unit dosage form.

Experiments with 3′-azido-3′-deoxythymidine suggest that a dose shouldbe administered to achieve peak plasma concentrations of the activecompound of from about 1 to about 75 μM, preferably about 2 to 50 μM,most preferably about 3 to about 30 μM. This may be achieved, forexample, by the intravenous injection of a 0.1 to 5% solution of theactive ingredient, optionally in saline, or orally administered as abolus containing about 1 to about 100 mg/kg of the active ingredient.Desirable blood levels may be maintained by a continuous infusion toprovide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusionscontaining about 0.4 to about 15 mg/kg of the active ingredient.

The uracil reductase inactivator may be administered in a dosage in therange of 0.01 to 50 mg per kilogram body weight of the recipient perday, preferably in the range of 0.01 to 10 mg per kilogram body weightper day, most preferably in the range of 0.01 to 0.4 mg per kilogrambody weight per day; an alternative preferred administration regime is0.5 to 10 mg/kg once per week.

The desired dose is preferably presented as one, two or more sub-dosesadministered at appropriate intervals throughout the day. Thesesub-doses may be administered in unit dosage forms for examplecontaining 1 to 200 mg, preferably 2 to 100 mg, most preferably 2 to 50mg of the uracil reductase inactivator.

Zidovudine and the uracil reductase inactivator are employed in anappropriate ratio whereby the above-mentioned toxic effects ofzidovudine are reduced or obviated without significant reduction of thetherapeutic effect of zidovudine; such a ratio (based on the respectiveweights of zidovudine and uracil reductase inactivator) is generally inthe range 1:1 to 1000:1, preferably in the range 5:1 to 500:1 andparticularly in the range 20:1 to 200:1.

Zidovudine and the uracil reductase inactivator are preferablyadministered in a pharmaceutical formulation, either in a singlepharmaceutical formulation containing both components or in separatepharmaceutical formulations each containing one of the components of thecombinations.

The present invention thus includes as a further feature apharmaceutical formulation comprising a uracil reductase inactivatoroptionally in combination with zidovudine or a pharmaceuticallyacceptable salt or ester thereof together with at least onepharmaceutically acceptable carrier or excipient.

Each carrier must be “pharmaceutically acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Formulations include those adapted for oral,rectal, nasal, topical (including buccal and sublingual), vaginal andparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. Such methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.

Formulations of the present invention adapted for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous (at pH10) or non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethylcellulose),lubricant, inert diluent, preservative, disintegrant (eg. sodium starchglycollate, cross-linked povidone, cross-linked sodiumcarboxymethylcellulose) surface-active or dispersins agent. Mouldedtablets may be made by moulding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide controlled release of the active ingredient therein using, forexample, hydroxypropylmethyl cellulose in varying proportions to providethe desired release profile.

Formulations for topical administration in the mouth include lozengescomprising the active ingredient in a flavoured basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulation for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams or spray formulations containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

Formulations for parenteral administration include aqueous (at pH 10)and non-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of an active ingredient.

The above-mentioned uracil reductase inactivators which are employed incombination with zidovudine in accordance with the present invention maybe prepared in conventional manner. For example, the inactivatorsreferred to above may be prepared by the methods described in J.Heterocycl. Chem. 19(3) 463-4 (1982) for the preparation of5-ethynyluracil; J. Chem. Soc. Perkin Trans. 1(16), 1665-70 (1981) forthe preparation of 5-(2-bromovinyl)uracil, 5-bromoethynyluracil and5-(2-bromo-1-chlorovinyl)uracil; Nucleic Acid Chemistry, Vol. 2, 927-30(1978) for the preparation 5-cyano-uracil; Nucleic Acids Research, 1(1)105-7 (1974) for the preparation of 5-vinyluracil; Z. Chem. 17(11)415-16 (1977) for the preparation of 5-trifluoromethyluracil; NucleicAcids Research 3 (10), 2845 (1976) for the preparation of5-(1-chlorovinyl)uracil.

The above nucleoside derivatives may also be prepared in conventionalmanner, for example in accordance with processes described in EuropeanPatent Specification No. 356166 for the preparation of 3′-fluoro-2′,3′-dideoxy-5-alkynyluridine compounds, such as2′,3′-dideoxy-5-ethynyl-3′-fluorouridine, and European PatentSpecification No. 272065 for the preparation of 5-alkynyluracilarabinosides, such as 1-(β-D-arabinofuranosyl)-5-prop-1-ynyluracil.

The novel 5-propynyluracil compound referred to above may be prepared byone of the following processes, namely:

a) treatment of a 5-propynyluridine compound to effect conversionthereof to the desired uracil compound; or

b) treatment of a uracil compound substituted in the 5-position by anappropriate leaving group with a propyne compound to give the desireduracil compound.

In the above process a), conversion may be effected by enzymatic means,for example by treatment of the uridine compound with a thymidinephosphorylase enzyme, advantageously in a buffered medium at a pH of 6to 8.

In the above process b), a uracil compound substituted in the 5-positionby a suitable leaving group e.g. iodo or bromo, is treated with a C₃₋₆alkyne in the presence of an appropriate palladium catalyst such as bis(triphenylphosphine) palladium (II) chloride and cuprous iodide in anamine solvent such as triethylamine.

Other 5-substituted uracil compounds for use in accordance with theinvention may be prepared in an analogous manner to those describedabove.

The following Examples illustrate the present invention.

EXAMPLE 1 5-Propynyluracil

A) To a stirred solution of 2′-deoxy-5-propynyluridine (European PatentSpecification No. 272065) (20 g, 75 mmol) in aqueous phosphate buffer atpH 6.84 (1250 mL) was added purified E. coli thymidine phosphorylase(10,000 units) (T. A. Krenitsky et al, Biochemistry, 20, 3615, 1981;U.S. Patent Specification No. 4,381,344) and alkaline phosphatase(10,000 units) [Sigma type VII-S from bovine intestinal mucosa] and thewhole mixture was incubated at 37° C. for 24 hours. The resulting whiteprecipitate was filtered, washed with water (3×100 mL), ethanol (2×100mL), ether (2×100 mL) and dried in vacuo over phosphorus pentoxide togive the title compound.

M.pt.: 275-280° C. (dec.)

¹H nmr δ (d₆DMSO) 11.5-11.0 (bs, 2H, NH), 7.61 (1H, s, H-6), 1.95 ppm(3H, s, CH₃)

Microanalysis calculated for C₇H₆N₂O₂: C,56.00; H,4.03; N,18.66

Found: C,55.92; H,4.05; N,18.77

B) 1-Arabinofuranosyl-5-propynyluracil, (2.92 g, 20.4 mmoles), 200 mlaqueous potassium phosphate, pH 6,8, 4,000 IU thymidine phosphorylase(Krenitsky, T. A. et al Biochemistry, 20,3615,1981 and U.S. Pat. No.4,381,444), 4,000 IU uridine phosphorylase (Krenitsky, T. A. et alBiochemistry, 20,3615,1981 and U.S. Pat. No. 4,381,444) and 2,000 IUalkaline phosphatase (Boehringer Mannheim) were stirred at 40° C. forfive days. Then 8,000 IU of thymidine phosphorylase, 20,000 IU uridinephosphorylase, 2,000 IU alkaline phosphatase and 30 IU acid phosphatase(Boehringer Mannheim) were added and incubation continued for anadditional five days. 5-Propynyluracil, being less soluble than thenucleoside, precipitated from the reaction mixture.

The precipitate and liquid were dried in vacuo, then 5-propynyluracilwas crystallized twice from hot water and vacuum dried at roomtemperature to give 0.92 g (6.1 mmoles) 5-propynyluracil in 59% yield.

¹H NMR δ (dDMSO) 11.2 ppm (bs, 2H, 1H and 3H), 7.6 ppm (1H, s, 6H), 1.95ppm (3H, s CH₃).

CHN calculated for C₇H₆N₂O₂: C, 56.00; H, 4.03; N, 18.66

Analyzed at: C, 55.95; H, 4.03; N, 18.60.

UV spectra: in 0.1 M HCl max at 287 nm and 231 nm; in 50 mM potassiumphosphate, pH 7.0 max at 287 nm and 231 nm; in 0.1 M NaOH max at 306 nmand 240 nm.

Mass spectrum gave peak at molecular ion weight of 151.

EXAMPLE 2 5-(Trimethylsilylethynyl)uracil

A solution of 5-iodouracil (8 g, 30 mmol) in redistilled triethylamine(500 mL) and dry DMF (10 mL) was degassed with oxygen-free nitrogen for15 minutes. Bis(triphenylphosphine)palladium (II) chloride (0.5 g),copper (I) iodide (0.5 g) and trimethylsilylacetylene (10 g, 102 mmol)were then added and the mixture was heated with stirring at 50° C. for24 hours. The cooled reaction mixture was filtered, the filtrateevaporated to dryness and the residue dissolved in dichloromethane (500mL). The organic solution was washed with a 2% aqueous solution ofdisodium EDTA (3×250 mL), water (3×200 mL), dried (Na₂SO4) andevaporated to dryness. The residue was triturated with ethanol to givethe first crop of the title compound. The solid filtered from thereaction mixture was also found to contain the required product but in amore impure form and so was worked up as above in a separate batch togive a second crop.

¹H nmr δ (d₆DMSO) 11.75-10.85 (2H, bs, NH), 7.75 (1H, s, H-6), 0.15 ppm(9H, m, SiCH₃).

EXAMPLE 3 5-Ethynyluracil

A solution of 5-(trimethylsilylethynyl)uracil (5.3 g, 25.4 mmol) in 0.2M solution of sodium methoxide in methanol (400 mL) was stirred at roomtemperature for 3 hours and neutralized to pH 7 with dilute hydrochloricacid. The precipitated product was filtered, washed with methanol anddried to give a first crop of the title compound. The filtrates andwashings were combined, evaporated to dryness and the residuecrystallised from methanol to give the second crop of product.Combination of both crops and a further recrystallisation from ethanolgave a pure product.

M.pt. : 260° C. (dec.)

¹H nmr δ (d₆DMSO) 11.6-10.8 (2H, bs, NH), 7.8 (1H, s, H-6), 4.03 ppm(1H, s, acetylenic H)

Microanalysis calculated for C₆H₄N₂O₂: C, 52.95; H, 2.96; N, 20.58

Found : C, 52.04; H, 2.92; N, 20.3

EXAMPLE 4

a) 2,4-Dimethoxy-5-iodo-pyrimidine

A dry IL round-bottomed flask was charged with 5-iodouracil (50 g, 0.21mol), phosphorus oxychloride (300 ml), and N,N-diethylaniline (6 drops).The heterogenous mixture was heated in a 120° C. oil bath under anitrogen atmosphere for 24 hours. The phosphorus oxychloride wasdistilled off (some product co-distills off). The reaction solution wasnext slowly and cautiously poured over ice (IL) and solid sodiumbicarbonate keeping the internal temperature at or below −20° C. (Thiswas accomplished by cooling in a dry-ice acetone bath). Once theaddition was complete, the reaction mixture was adjusted to pH 7 byaddition of solid sodium bicarbonate. The mixture was extracted withmethylene chloride and the organic fractions dried by passage throughphase separator paper. The crude solution of2,4-dichloro-5-iodopyrimidine was immediately added dropwise to asolution containing MeOH (400 ml) and sodium methoxide (28.8 g, 0.533mol). This addition took 1 hour. The reaction was then stirred at roomtemperature overnight. The solution was neutralized with CO₂(gas),extracted with methylene chloride, dried over anhydrous Na₂SO₄ filteredand concentrated. The crude product was adsorbed onto silica gel (100 g)and loaded onto a 400 g silica gel flash chromatography column. Thecolumn was eluted with 90:10 hexanes: ethyl acetate (v:v). Theappropriate fractions were combined and concentrated to a white solid asthe title compound.

Yield 26.7 g (48%)

200 MHZ NMR CDCl₃δ=3.97 (s, 3H); 4.02 (s, 3H), 8.43 (s, 1H).

b) 2,4-Dimethoxy-5-(β-trimethylsilyl)-ethynylpyrimidine

A dry 1 L round-bottomed flask under a nitrogen atmosphere was chargedwith the product of stage a) (26.7 g, 0.10 mol), dry methylene chloride(Aldrich, 150 mL), dry Et₃N (freshly distilled from KOH pellets, 250mL). The system was evacuated and purged with nitrogen several times viaa Firestone valve. Trimethyl-silylacetylene (21.2 mL, 0.15 mol; Aldrich)was added by syringe. Next were added bis(triphenylphosphine)palladium(II) chloride (Aldrich 5.84 g, 8.32 mmol) and copper (I) iodide (Aldrich4.76 g, 25 mmol). The mixture was heated in a 60° C. oil bath for 2hours, cooled and filtered through Celite. The filtrate was concentratedin vacuo. The residue was diluted with toluene (100 mL) and then thetoluene was removed in vacuo. The residue was taken up into methylenechloride (200 mL), filtered and the filtrate extracted with 5% aq.ethylenediaminetetraacetic acid, disodium salt dihydrate (3×100 mLAldrich), H₂O (1×100 mL). The organic layer was dried via passagethrough phase separator paper and concentrated in vacuo. The product waspurified on a Waters Prep 500 eluting with 95:5 hexanes: ethyl acetate(v:v). The crude product was adsorbed onto 100 g of silica gel andloaded onto a 400 g silica gel flash chromatography column. The columnwas eluted with 97.5:2.5 hexanes: ethyl acetate (v:v). The appropriatefractions were combined and concentrated.

Yield 16.94 g (73%).

A 1.2 g sample of the resulting compound was bound to 6 g of silica geland loaded onto a 50 g flash chromatography column. The column waseluted with hexanes: ethyl acetate 95:5 (v:v). The appropriate fractionswere combined, concentrated, stripped with CH₂Cl₂ (2×30 mL), and driedin vacuo to yield 1.000 g of the title compound, m.p. 72.5-73° C. Lit.m.p. 73-74° C. J. Heterocyclic Chem., 19, 463 (1982).

c) 5-(β-trimethylsilyl)ethynyluracil

A dry 3-necked round-bottomed flask under nitrogen was charged with2,4-dimethoxy-5-(β-trimethylsilyl)ethynylpyrimidine (6.5 g, 27.5 mmol),dry acetronitrile (120 mL Aldrich), sodium iodide (oven dried in vacuo80° C., 18 h, 12.4 g, 82.7 mmol) and chlorotrimethylsilane (10.5 mL,82.7 mmol freshly distilled). The mixture was heated at reflux for 3hours and then concentrated in vacuo. The residue was digested with asolution containing methanol (40 mL) and water (20 mL) and the productfiltered off to give 1.48 g (26%). The product was dissolved inchloroform and the solution adsorbed onto silica gel 7 g) which was thenloaded onto a 35 g silica gel flash chromotography column. Elution withchloroform:methanol 95:5 (v:v) followed by chloroform:methanol 90:10(v:v) and evaporation of the product-containing fractions yielded 1.23 gof the title compound as a white solid.

d) 5-Ethynyluracil

A solution containing 5-(β-trimethylsilyl)ethynyluracil (3.85 g, 18.4mmol) and methanol (370 mL) was treated with a second solutioncontaining sodium hydroxide (2.3 g, 57.5 mmol) and water (18 mL). Themixture was stirred at room temperature for 2 hours and thenconcentrated in vacuo. The residue was suspended in water (35 mL) andthe pH adjusted to 5 using 0.1 N HCl. The solids dissolved and then asecond precipitate formed when the pH-5. The product was filtered,washed with H₂O, and then dried in vacuo to give 2.3 g (92%) of5-ethynyluracil as a light beige powder.

Microanalysis calculated for C₆H₄N₂O₂: C, 52.95: H, 2.96; N, 20.58

Found: C, 52.79; H, 3.02; N, 20.44

EXAMPLE 5

a) 2′, 3′-5′-Tri-O-Acetyl-5-iodouridine

A dry 250 mL round-bottomed flask was charged with 5-iodouridine (10 g,27 mmol Aldrich), anhydrous pyridine (30 mL) and acetic anhydride (30mL). The reaction was stirred at room temperature for 30 minutes under anitrogen atmosphere and the solvent removed in vacuo. The compound wasdiluted with toluene (2×50 mL) and the toluene removed in vacuo. Theproduct was purified on a 75 g flash chromatrography column which waseluted with 90:10 (v:v) CHCl₃:MeOH. The appropriate fractions werecombined and concentrated to give the title compound as a white foam.This was used directly in the next stage.

b) 2′,3′,5′-Tri-O-Acetyl-5-[2-(trimethylsilyl)ethynyl]uridine

A dry 1 L round-bottomed flask equipped with a reflux condenser (underN₂ atmosphere) was charged with the product of stage a) (27 mmol), drymethylene chloride (260 mL, Aldrich) and dry triethylamine (260 mL,freshly distilled from NaOH pellets). The system was evacuated andpurged with nitrogen several times and remained under a nitrogenatmosphere. Next was added (trimethyl-silyl)acetylene (11.65 mL, 82mmol; Aldrich) followed by copper (I) iodide (Aldrich, 1.57 g, 8.2 mmol)and bis(triphenylphosphine)palladium II chloride (Aldrich, 1.85 g, 2.6mmol). The mixture was heated in a 60° C. oil bath for 30 minutes,cooled, and filtered. The filtrate was concentrated in vacuo. Theresidue was taken up into CH₂Cl₂ (300 mL), filtered, washed with 5% aq.ethylenediaminetetraacetic acid, disodium salt (2×75 mL), H₂O (100 mL),dried over Na₂SO₄, filtered and concentrated in vacuo.

The resulting compound was bound to 50 g of silica gel and loaded onto a400 g silica gel flash chromatrography column which was eluted withCHCl₃. The product fractions were combined and concentrated to yield thetitle compound as light yellow foam.

Yield 13 g

300 MHz NMR CDCl₃ δ 8.2 (br s, NH, 1H), 7.77 (s, 1H, H6), 6.11 (d, H1′,1H), 2.22 (s, 3H, OAc), 2.13 (s, 3H, OAc), 2.11 (s, 3H, OAc), 0.22 (s,9H, SiMe₃).

c) 5-Ethynyluridine

The product of stage b) (9.5 g, 24 mmol) was dissolved in methanol (200mL) and diluted with a solution containing sodium (0.8 g) and methanol(100 mL). The reaction was stirred at room temperature for 2 hours andwas then neutralized using Dowex 50W-X8 (H⁺ form) resin. The resin wasremoved by filtering and washed with methanol. The filtrate wasconcentrated in vacuo to give 4.85 g of a beige solid. The compound waspurified on a Waters Prep 500 reverse phase C₁₈ column which was elutedwith H₂O/MeOH 85:15 (v:v) to give 1.2 g of the title product (whitesolid). Impure fractions were re-chromatographed. An additional 1.94 gof product were obtained.

Yield 49%

Calculated: % C,49.25 % H,4.47 % N,10.44

Found: % C,49.07 % H,4.53 % N,10.32

200 MHz NMR (DMSOd₆) δ 11.60 (br s, NH, 1H), 8.36 (s, H6, 1H), 5.72 (d,J=4.3 Hz H1′, 1H), 4.01 (s, 1H, C═C—H).

The following Examples illustrate pharmaceutical formulations in whichthe “Active Ingredient” is 5-propynyluracil.

EXAMPLE 6 Tablet Formulations

The following formulations 6A, 6B and 6C are prepared by wet granulationof the ingredients (except the magnesium stearate) with a solution ofthe povidone followed by drying of the granules, addition of themagnesium stearate and compression.

mg/tablet mg/tablet Formulation 6A Active ingredient 5 2 Lactose, B.P.205 75 Povidone, B.P. 15 10 Sodium starch glycollate 20 10 Magnesiumstearate 5 3 250 100 Formulation 6B Active ingredient 5 2 Lactose, B.P.155 — Avicel PH 101 50 25 Povidone, B.P. 15 10 Sodium starch glycollate20 10 Magnesium stearate 5 3 250 50 Formulation 6C Active ingredient 5Lactose, B.P. 205 Starch 50 Povidone, B.P. 6 Magnesium stearate 4 270

The following formulation 6D is prepared by direct compression of theadmixed ingredients. The lactose used is of the direct compression type.

Formulation 6D mg/tablet Active ingredient  5 Lactose 155 Avicel PH 101100 260

The following formulation 6E is a controlled release tablet and isprepared by wet granulation of the ingredients (except magnesiumstearate) with a solution of the povidone, followed by drying of thegranules, addition of the magnesium stearate and compression.

Formulation 6E mg/tablet Active ingredient 5Hydroxypropylmethylcellulose 110 (Methocel K4M Premium) Lactose, B.P. 50Povidone, B.P. 28 Magnesium stearate 7 200

EXAMPLE 7 Capsule Formulations

The following formulations 7A and 7B are prepared by admixing theuncompressed ingredients and filling into a two-part hard gelatincapsule.

mg/capsule Formulation 7A Active ingredient 10 Lactose, B.P. 250 Sodiumstarch glycollate 25 Magnesium stearate 5 290 Formulation 7B Activeingredient 5 Pregelatinized starch NF15 245 250 Formulation 7C Activeingredient 10 Macrogol 4000, B.P. 340 350

The Macrogol 4000, B. P. is melted and the active ingredient dispersedtherein. The thoroughly mixed melt is then filled into a two-part hardgelatin capsule.

EXAMPLE 8

Injectable Formulation Active ingredient 10 mg Sterile, pyrogen freePyrophosphate buffer (pH 10), q.s. to 10 ml

The active ingredient is dissolved in most of the pyrophosphate buffer(35-40° C.), then made up to volume and filtered through a sterilemicropore filter into a 10 ml amber glass vial (type 1) and sealed witha sterile closure and overseal.

EXAMPLE 9

Suppository Formulation mg/suppository Active ingredient, 63 μm*  10Hard fat, B.P. (Witepsol H15- 1790 Dynamit Nobel) 1800 *The activeingredient is used as a powder wherein at least 90% of the particles areof 63 μm or less.

Our-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C. maximum. The active ingredient is sifted through a 200 Mm sieve andadded to the molten base with mixing, using a silverson fitted with acutting head, until a smooth dispersion is achieved. Maintaining themixture at 45° C., the remaining Witepsol H15 is added to the suspensionand stirred to ensure a homogeneous mix. The entire suspension is passedthrough a 250 μm stainless steel screen and, with continuous stirring,is allowed to cool to about 40° C. At a temperature of 38° C. to 40° C.1.80 g of the mixture is filled into suitable plastic moulds. Thesuppositories are allowed to cool to room temperature.

Determination of Uracil Reductase Inactivation

Uracil reductase (1 μM) (dihydropyrimidine dehydrogenase, EC 1.3.1.2)purified from bovine liver was incubated with 100 μM inactivator and 5mM dithiothreitol (enzyme reductant) at 37 for 30 minutes in 0.05 MTris-HCl at pH 8.0. The enzyme and inactivator were diluted 100-foldinto the assay buffer, which contained 200 μM NADPH, 200 μM thymine and1 mM dithiothreitol in Tris-HCl at pH 8.0. The velocity of the enzymewas determined spectrophotometrically. These velocities have beencorrected for NADPH oxidase activity, which was less than 10% of therate of thymine-dependent oxidation of NADPH. The % inactivation of theenzyme was equal to 100% minus the percent of enzymatic activityremaining. Enzyme incubated without inhibitor was stable under theseconditions. Parenthetical values are the relative first-order rateconstants for inactivation of enzyme determined from similar experimentswhere the fractional activity was measured as a function of the time ofincubation of 50 μM inactivator with enzyme.

The results are given below:

Compound % Inactivation 5-ethynyluracil 100 (100) 5-cyanouracil^(a) 100(14) 5-propynyluracil 100 (8) 5-bromoethynyluracil 100 (8)5-(1-chlorovinyl)uracil 100 (5) 5-iodouracil 100 (4) 5-bromovinyluracil 93 5-hex-1-ynyluracil^(a)  90 5-vinyluracil^(a,b)  86 5-trifluorouracil 75 5-bromouracil  75 5-(2-bromo-1-chlorovinyl)uracil  68 ^(a)Theinhibition was reversible since enzyme treated with this derivativeslowly regained activity after a 100-fold dilution into the assaymixture. ^(b)These nucleobases were generated in situ by treating therespective nucleosides with 40 units/ml of thymidine phosphorylase in 35mM potassium phosphate for 20 minutes prior to addition to uracilreductase. The parent nucleosides were not inactivators.

Protection from Zidovudine Toxicity

Male mice were doses p.o. with 1000 mg/kg/day of zidovudine for 30 dayseither alone, or 1.5 hours after dosing with 2 mg/kg/day of5-ethynyluracil (5-EU). Other groups of mice (a) were dosed with 2mg/kg/day of 5-ethynyluracil alone; and (b) served as controls,receiving neither zidovudine nor 5-ethynyluracil. Levels of haematocrit,haemoglobin and red blood cells were determined.

The results are as follows:

RED BLOOD HEMATOCRIT HEMOGLOBIN CELLS GROUP (%) (g/dl) (million/ml)Control 48.1 16.0 9.7 5-EU 45.9 15.1 9.1 Zidovudine 34.5 11.2 6.1Zidovudine 43.1 13.9 7.6 plus 5-EU

What is claimed is:
 1. A pharmaceutical composition comprising anucleoside derivative having a cleavable sugar portion wherein saidcleavable sugar portion is 2′, 3′-dideoxyribose or a derivative thereofhaving a 2′ or 3′ substituent, which generates in vivo uracilsubstituted in the 5-position by: C₂₋₆ alkynyl; and at least onepharmaceutically acceptable carrier.
 2. The composition of claim 1wherein the 2′- or 3′-substituent is halo.
 3. The composition of claim 1wherein the 2′- or 3′-substituent is fluoro.
 4. The composition of claim1 wherein the compound is a nucleoside derivative of 5-propynyluracil or5-ethynyluracil.
 5. The composition of claim 1 comprising2′,3′-dideoxy-5-ethynyl-3′-fluorouridine.
 6. The composition of claim 1which is suitable for oral administration.
 7. The composition of claim 1which is in the form of a tablet or capsule.
 8. The composition of claim1, wherein the uracil generated in vivo is 5-ethynyluracil.
 9. Thecomposition of claim 1, wherein the uracil generated in vivo is5-propynyluracil.